Chlamydia trachomatis is the causative agent of several human diseases including trachoma, the leading cause of infectious blindness worldwide, and is the most common form of sexually transmitted disease in the U.S. and developed countries. Chlamydiae are obligate intracellular bacteria that undergo their life cycles entirely within an intracellular vesicle that is not acidified and does not fuse with lysosomes. The chlamydial inclusion seems to be isolated from established routes of intracellular trafficking. Other than vacuoles containing other chlamydiae, no cellular vesicles are known to fuse with the chlamydial inclusion. The inclusion membrane grows throughout the developmental cycle to contain the multiplying bacteria yet the biosynthetic source of the inclusion membrane is unknown. Although chlamydiae obviously acquire essential nutrients from the host cell, the mechanisms for obtaining these across the inclusion membrane are unknown. We have employed a variety of specific probes for various cellular organelles in conjunction with conventional fluorescence and confocal microscopy in an attempt to identify cellular organelles that may interact with the chlamydial inclusion. Our results indicate a direct involvement of the Golgi apparatus in trafficking of sphingolipids to the chlamydial inclusion and imply a close interaction between the chlamydial inclusion and the Golgi network. Modification of the inclusion membrane by insertion of chlamydial proteins has been demonstrated by a novel experimental approach that detected proteins specific to the infected cells but not on purified chlamydiae. The first of what appear to be multiple parasite-specified polypeptides in the inclusion membrane has been cloned, sequenced, and expressed. Collectively, these data identify a chlamydial protein that is released from intracellular chlamydiae to modify the inclusion membrane. This modification is likely essential for the successful multiplication of the parasite.